luciferase crpv igr renilla luciferase sequence  (Addgene inc)

Journal: PLOS One

Article Title: Cnot4 heterozygosity attenuates high fat diet-induced obesity in mice and impairs PPARγ-mediated adipocyte differentiation

doi: 10.1371/journal.pone.0316417

Figure Lengend Snippet: γ . (A) Protein expression of PPARγ in MEFs. (B) mRNA levels in MEFs at 6 hours after 1 μM rosiglitazone treatment (n = 3). (C) Reporter assay using the luciferase reporter plasmid harboring PPARγ response element (PPRE). The reporter plasmid was transfected into MEFs at 24 hours before treatment of 1 μM rosiglitazone, and cells were harvested 6 hours after rosiglitazone treatment (n = 3). (D) Western blot of PPARγ in cytoplasm (Cyto) and nuclear (Nuc) fraction of MEFs. (E) ChIP-qPCR for PPARγ-bound aP2 promoter regions in MEFs (n = 2). All values are means ± SEM. One-way ANOVA with Sidak’s multiple-comparisons test (B and C). Numbers above square brackets show significant P -values.

Article Snippet: MEFs were seeded in 24-well plates (3.0 x 10 4 cells/well), and cells were transfected with 500 ng of reporter plasmid DNA containing PPARγ response element following luciferase sequence (plasmid no. 1015; Addgene) using Lipofectamine 3000 (Invitrogen) at 24 hours after plating.

Techniques: Expressing, Reporter Assay, Luciferase, Plasmid Preparation, Transfection, Western Blot, ChIP-qPCR

Journal: Nature Communications

Article Title: Auricular malformations are driven by copy number variations in a hierarchical enhancer cluster and a dominant enhancer recapitulates human pathogenesis

doi: 10.1038/s41467-025-59735-w

Figure Lengend Snippet: a Epigenetic profiling at the BCE core locus in early (D11) and late hCNCCs (P4). Previously reported mouse 594 bp ECR region resides in the mEC1 homologous sequence. b Schematic of hESCs differentiation to PA-like hCNCCs: The process involves transitioning H9 cell line hESCs to early hCNCCs using NCC induction (NCCI) medium, followed by BMP2/ChIR treatment to reach late hCNCCs stage, and finally achieving PA-like hCNCCs with retinoic acid (RA) introduction. c HMX1 expression analysis across four cell states, presented as mean ± S.E.M. ( n = 6). P -values were calculated using unpaired Student’s t -test (two-tailed). d, e Luciferase assays evaluating candidate enhancers at the BCE core locus in hESC ( d ), and PA-like hCNCC ( e ), including SV40 enhancer (positive control) and empty vector (negative control). Results from three independent experiments, each with four technical replicates ( n = 12), are shown. The box plot displays the median (center line), 25th-75th percentiles (box bounds), and whiskers extending to 1.5×interquartile range from each quartile (10th-90th percentiles shown as reference), with outliers plotted individually. f CRISPR/Cas9 strategy to delete two active enhancers (hEC1 and hEC2, ~9 kb), and gene expression comparison across four cell states in WT and knockout cell lines ( n = 3). P -values were calculated using unpaired Student’s t -test (two-tailed). The boxplot definition is same as d, e . Source data are provided as a Source Data file.

Article Snippet: The transfection mix included 0.5 μg of pGL3-enhancer plasmids with firefly luciferase gene sequences driven by an SV40 promoter (Promega, catalog no. E1761), 0.02 μg of pRL-CMV Renilla luciferase (Promega, catalog no. E2261) as an internal control, and 1.5 μL of FuGENE HD (Promega, catalog no. E2691), all diluted in Opti-MEM I Reduced-Serum Medium (Gibco, catalog no. 31985062).

Techniques: Sequencing, Expressing, Two Tailed Test, Luciferase, Positive Control, Plasmid Preparation, Negative Control, CRISPR, Gene Expression, Comparison, Knock-Out